Extracellular histones are major mediators of death in sepsis

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1 SUPPLEMENTARY MATERIALS Extracellular histones are major mediators of death in sepsis Jun Xu 1, Xiaomei Zhang 2, Rosana Pelayo 3, Marc Monestier 4, Concetta T. Ammollo 1, Fabrizio Semeraro 1, Fletcher B. Taylor 1, Naomi Esmon 1, Florea Lupu 1 and Charles T. Esmon 1,2 1 Cardiovascular Biology Research Program, 2 Howard Hughes Medical Institute, Oklahoma City, OK 73104, 3 Immunology and Cancer Research Program, Oklahoma Medical Research Foundation, 4 Temple Autoimmunity Center, Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, PA 19140, USA Methods Identification of proteins from stimulated macrophages RAW264.7 cells were stimulated with 1 µg ml 1 LPS and 20 ng ml 1 interferon gamma (IFN) for 24 h, washed with PBS, and cultured in Opti MEM medium (Invitrogen) with or without 100 nm human APC for 24 h. The conditioned medium was filtered through a 0.22 µm filter and concentrated 80 fold with an Amicon Ultra 10,000 (Millipore). Protein bands were electrotransferred onto PVDF membrane (Immobilon P, Millipore) after SDS PAGE, stained with GelCode Blue (PIERCE) and sequenced by Edman degradation (Applied Biosystems). Ca 2+ influx imaging EA.hy926 cells were seeded on glass bottom dishes (MatTek Corp.) and pre loaded with Fluo 4AM (Molecular Probes) according to Manufacturer s instruction. After washing, cells were incubated with HBSS containing 3 mm CaCl 2 and basal [Ca 2+ ]i for regions of interest was recorded before the cells were treated with 20 µg ml 1 H4 in the presence or absence of antibody to H4 (100 µg ml 1 ). Fifty images were collected at 5 s intervals in time series using a Nikon C1 confocal microscope and analyzed with EZ C1 (Nikon) and ImageJ (NIH) software. 21

2 Supplemental Figure 1 Identification of extracellular histones cleaved by APC. Activated mouse macrophage cells (RAW264.7) stimulated by LPS (1µg ml 1 ) and IFN (20 ng ml 1 ) for 24 h were cultured in Opti MEM medium with or without 100 nm human APC for another 24 h. Concentrated conditioned medium was either (a) measured for its cytotoxicity toward EA.hy926 after 1 h by flow cytometry for PI staining or (b) subjected to SDS PAGE and Coomassie blue staining or (c) subjected to SDS PAGE and Western blotting for histone H3. 22

3 Supplemental Figure 2 (a) HUVEC +Histones +H1 +H2A +H2B +H3 +H4 PI (b) HUVEC +Histones +APC+histones PI Cytotoxicity of extracellular histones toward HUVEC and APC inhibition of histone cytotoxicity. (a) HUVEC were cultured with calf thymus histones (50µg ml 1 ) or calf thymus histone H1, H2A, H2B, H3 or H4 (20µg ml 1 ) for 1 h at 37ºC. Cell damage was measured by flow cytometry for PI staining. (b) APC (100 nm) was absent or present during the incubation with histones in the above assays. 23

4 Supplemental Figure 3 Dynamic changes of calcium influx in EA.hy926 cells treated with H4. The montage shows the first (pretreatment) and last (post treatment) image of representative time lapse recordings (50 frames at 5 second intervals) of cells loaded with calcium indicator dye and treated with histone H4 in the absence (a c) or presence (d e) of antibody to H4. (c and f) Time course integrations of mean fluorescence intensity of 20 cells. 24

5 Supplemental Figure 4 Intravascular histones induce pathological landmarks of acute lung injury: microhemorrhage, intravascular thrombosis and capillary leak. (a b) disruption of microvascular integrity and intra alveolar hemorrhage, as illustrated by the massive presence of red blood cells (b) within the alveolar space, as compared to the normal structural aspect (a) of non challenged animals. (c e) Double immunofluorescence staining for fibrin (c) and a platelet marker (P selectin, d) demonstrate the widespread 25

6 presence of fibrin (c) and platelet rich (d) thrombi. (e) PTHA staining of fibrin exudate within the alveolar space. (f g) Electron micrographs showing a platelet rich microthombus (Th) in the alveolar capillary microvasculature (g, higher magnification inset), as well as fibrin (fn) and collagen (coll) accumulation within the inter alveolar septum. av, alveolae; B, bronchiolae; cap, alveolar capillaries. Magnification bars: a and b, 500 µm; c and d, 50 µm; e, 100 µm; f and g, 1 µm. 26